CSNK2A1-mediated MAX phosphorylation upregulates HMGB1 and IL-6 expression in cholangiocarcinoma progression

Background: We established a novel diethylnitrosamine (DEN) -induced mouse model that reflected the progression of cholangiocarcinoma (CCA) from atypical cystic hyperplasia. Methods: BALB/c mice were administered DEN by oral gavage. Cells isolated from livers were analyzed for expression of CSNK2A1, MAX and MAX-interacting proteins. Human CCA cell lines (MzChA-1, HuCCT1), normal human cholangiocyte (H69), human hepatic stellate cells (LX-2), macrophages (RAW 264.7), and primary hepatic cells were used for cellular and molecular biology assays. Results: Expression of MAX, CSNK2A1, C-MYC, β-catenin, HMGB1, and IL-6 was upregulated in hepatic cells from CCA liver tissue. The half-life of MAX is higher in CCA cells, and this favors their proliferation. Overexpression of MAX increased growth, migration, and invasion of MzChA-1, whereas silencing of MAX had the opposite effect. MAX positively regulated IL-6 and HMGB1 through paracrine signaling in HepG2, LX2, and RAW cells and autocrine signaling in MzChA-1 cells. CSNK2A1-mediated MAX phosphorylation shifts MAX-MAX homodimer to C-MYC-MAX and β-catenin-MAX heterodimers and increases the HMGB1 and IL-6 promoter activities. Increase of MAX phosphorylation promotes cell proliferation, migration, invasion, and cholangiocarcinogenesis. The casein kinase 2 inhibitor CX-4945 induces cell cycle arrest and inhibits cell proliferation, migration, invasion, and carcinogenesis in MzChA-1 cells through the downregulation of CSNK2A1, MAX, and MAX-interaction proteins. Conclusion: C-MYC-MAX and β-catenin-MAX binding to E-box site or β-catenin-MAX bound to TCFs/LEF1 enhanced HMGB1 or IL-6 promoter activities, respectively. IL-6 and HMGB1 secreted by hepatocytes, HSCs, and KCs exert paracrine effects on cholangiocytes to promote cell growth, migration, and invasion and lead to the progression of cholangiocarcinogenesis. CX-4945 provides perspectives on therapeutic strategies to attenuate progression from atypical cystic hyperplasia to cholangiocarcinogenesis.


INTRODUCTION
Cholangiocarcinoma (CCA) is a common invasive cancer of the intrahepatic and extrahepatic bile ducts. According to the anatomical location, it is divided into 3 subtypes: intrahepatic CCA (iCCA), perihilar CCA, and distal CCA. [1] Patients not suitable for surgery receive systemic chemotherapy with gemcitabine and cisplatin in addition to standard care and have a median overall survival of <1 year. [2,3] Recent research has shown that checkpoint inhibitors may be an emerging cornerstone to CCA therapy. [4] Animal models of CCA allow us to study both the pathobiology of disease and treatment responses. [5] The chemotoxic-induced model, responsible for inducing genotoxicity and promoting CCA formation, is a classic animal model of CCA. [6,7] These chemotoxic substances include diethylnitrosamine (DEN) and N-nitrosodimethylamine (NDMA), nitrosamine, furan, thioacetamide (TAA), and carbon tetrachloride (CCL4). [5,6] Previously, we reported that chronic cholestasis accelerated DENmediated CCA formation. [5] Here, we established a novel mouse model recapitulating the progression from atypical cystic hyperplasia to CCA with DEN treatment.
Protein kinase CK2 (known as CSNK2) is a highly conserved serine/threonine kinase that regulates cell proliferation, cell cycle progression, invasiveness, and tumorigenesis. [8] CSNK2α1 (CSNK2A1 and CK2α) plays a crucial role in cancer progression through MYC and Wnt/β-catenin pathways. [9] Furthermore, CSNK2A1 can also induce phosphorylation of various molecules. [10] β-catenin plays a crucial role in tumorigenesis as an intracellular signaling molecule in the WNT signaling pathway. [11] High-mobility group box-1 (HMGB1) regulates apoptosis, autophagy, and gene transcription and is a critical protein in the pathogenesis of acute liver injury and chronic liver diseases. [12,13] HMGB1 knockdown has been shown to inhibit proliferation and promote apoptosis and autophagy in CCA cell lines HuB28 and HuCCT1. [14] It is established that proinflammatory cytokines, such as IL-6, enhance the pathogenesis of chronic inflammation-induced CCA. IL-6 released by cancer-associated fibroblasts has been shown to promote carcinogenesis. [15] Elevated IL-6 has been considered a poor prognostic marker in patients with CCA. [15] C-MYC is a basic helix-loop-helix leucine zipper transcription factor that forms a heterodimeric complex with MAX (MYC-associated factor X) and binds with the E-box sequence to activate transcription of target genes. [5] We previously reported the importance of C-MYC in the murine model of CCA. [5] It has been established that MAX acts as a tumor suppressor and rewires metabolism in small-cell lung cancer. [16] Paradoxically, MAX deletion destabilizes MYC protein and abrogates Eµ-MYC lymphoma development. [17] The role of MAX in CCA development remains largely unknown.
We developed a novel mouse model that progresses from atypical cystic hyperplasia to CCA. Compared with previously reported models, this model has a shorter CCA development time, more evident pathological manifestations, and a closer resemblance to the development of CCA in humans. Furthermore, using this model, we found that MAX and MAX-interacting proteins are targetable master regulators of CCA progression.

Additional methods
All other methods used are described in detail in the Supplemental Methods section of the Supplemental Materials.

Establishment of a novel model of cholangiocarcinogenesis
Some chemically induced CCA models are suboptimal due to high mortality, low incidence, and the need for surgical procedures (Supplemental Table S1, http://links.lww.com/ HC9/A362). [1,5] At first, the effects of DEN treatment were assessed on biliary proliferation and development of atypical cystic hyperplasia, cholangiomas, and CCA. The intrahepatic bile ducts were confined to the portal space at week 1 ( Figure 1A; Supplemental Figure S1A, http://links.lww. com/HC9/A350). Proliferating cholangiocytes formed a welldefined lumen at week 3 and resembled bile duct ligation at day 3 ( Figure 1A and Supplemental Figure S1B, http://links. lww.com/HC9/A350). Biliary proliferation increased rapidly from weeks 3 to 6 ( Figure 1A; Supplemental Figure S1C, http://links.lww.com/HC9/A350). Atypical cystic hyperplasia appeared 9 weeks after the administration of DEN. The proliferation of intrahepatic bile ducts extended to the periportal area along the sinusoid and adjacent liver parenchyma ( Figure 1A; Supplemental Figure S2A, http:// links.lww.com/HC9/A351) and instead were associated with inflammatory cell infiltration (Supplemental Figure S2B, http://links.lww.com/HC9/A351). The papillary proliferation of the atypical biliary epithelium showed multilayering of the nuclei, loss of cell polarity, and nuclear hyperchromasia within the bile ducts at week 13 ( Figure 1A). Sinusoidal invasion and portal vein invasion are considered the most frequent modes of iCCA spread in humans; in our mouse model, we also found that tumor cells invaded sinusoids (Supplemental Figure S2C, http://links.lww.com/HC9/A351) and portal veins (Supplemental Figure S2D, http://links.lww. com/HC9/A351) at week 13. These pathological features demonstrate that the DEN-treated mouse model mimics human CCA progression well and is therefore an attractive model for studying biliary proliferation, atypical cystic hyperplasia, cholangiomas, and CCA progression.
Mice treated with DEN had 16% mortality at 11 and 13 weeks ( Figure 1B). Significantly higher liver/body weight ratio ( Figure 1C) and lower overall body weight ( Figure 1D) were observed in the DEN-fed mice compared with control mice beginning 6 weeks after treatment. Significantly higher liver weights were found in the DEN-fed mice than in the control mice at week 13 ( Figure 1E).
We examined changes in the hepatobiliary system over time after DEN treatment and found that bilirubin levels in mice treated with DEN increased sharply at week 3 and remained high up to week 13 ( Figure 1F). An acute increase in alanine transaminase (ALT) and aspartate transaminase (AST) levels occurred after 1 week of oral gavage with DEN and steadily increased thereafter ( Figure 1G, H).
CK19 was used to measure cholangiocyte proliferation. We found proliferating cholangiocytes (CK19 positive) at week 1 that increased dramatically from weeks 3 to 9 and plateaued around weeks 11 and 13 in the DEN group ( Figure 1I; Supplemental Figure S3A, http://links.lww.com/ HC9/A352). Hepatic macrophages are often referred to as KCs, since KCs represent the main fraction of liver macrophages. Mouse KCs are identified by F4/80 staining. We observed a significant increase in the number of F4/80-positive cells in mice livers treated with DEN at week 1, and levels continued to increase up to week 13 ( Figure 1J; Supplemental Figure S3B, http://links.lww.com/ HC9/A352).
MAX expression and effect on cell growth, migration, invasion, and tumorigenesis MAX has both favorable and unfavorable consequences in tumorigenesis. [16,[18][19][20] We focused on MAX because we found higher human MAX protein and mRNA levels in PSC and CCA compared with normal liver, especially in the nucleus of human and mouse CCA (Figure 2A, B and Supplemental Figure S7A, B, http://links.lww.com/HC9/ A356). MAX mRNA levels are higher in iCCA compared with adjacent nontumor liver tissue (GSE76297) (Supplemental Figure S8A, http://links.lww.com/HC9/A357) and iCCA compared with normal human biliary epithelial cells (GSE32225) (Supplemental Figure S8B, http://links. lww.com/HC9/A357). Moreover, the GEPIA database shows that MAX mRNA significantly increases in CCA compared with normal liver tissues (Supplemental Figure  S8C, http://links.lww.com/HC9/A357). MAX protein levels in the cytoplasm and nucleus were higher in human CCA compared with normal liver tissues ( Figure 2C). One possible explanation is that MAX affects cellular stability.
To investigate this possibility, we performed a chase assay using cycloheximide and observed that the half-life of MAX was~24 hours in MzChA-1 cells, 48 hours in cholangiocytes isolated from mouse liver with CCA, and 12 hours in cholangiocytes isolated from control mice ( Figure 2D, E). Therefore, the stability of MAX from normal cholangiocytes was lower than that of MzChA-1and CCA-derived cholangiocytes ( Figure 2E). The enhanced stability of MAX in cancer cells led us to examine whether it regulated cell proliferation. Compared with scramble siRNA, MAX siRNA inhibited MAX protein by 69% ( Figure 2F). In contrast, overexpression of MAX led to a 2.2-fold increase in its protein level ( Figure 2F). We found that silencing MAX suppressed MzChA-1 migration ( Figure 2G), cell growth ( Figure 2H), and invasion ( Figure 2I). MAX knockdown of MzChA-1 cells increased G1 fraction and decreased the G2 fraction relative to cells transduced with control siRNA ( Figure 2J, K), indicating inhibited cell cycle progression. MAX overexpression in MzChA-1 cells decreased G1 fraction and increased the G2 fraction relative to cells transduced with empty vector ( Figure 2J, K), indicating promoted cell cycle progression. Overexpression of MAX had a profound promotion of anchorage-independent growth, whereas MAX silencing has the opposite effect (Supplemental Figure S9, http://links.lww.com/HC9/A358).
To confirm their effects in vivo, we established MzChA-1 cell lines that either stably overexpress MAX or have reduced expression of MAX using CRISPR as described [21] ( Figure 2L). These cells were implanted into the left lobe of the liver, and their growth was examined in this orthotopic model. Figure 2M (top) shows the tumor at the site of injection after 30 days. CCA cells overexpressing MAX resulted in much larger tumor sizes as compared with respective controls. In contrast, CCA cells overexpressing CRISPR targeting MAX had much smaller tumor sizes ( Figure 2M). Figure 2M (middle) shows H&E staining of the tumors and the aggressive histological features of the MAX overexpressing or the MAX knocked-down CCAs. Consistently, PCNA staining is highest for tumor overexpressing MAX, and the opposite is true for MAX knockdown ( Figure 2M, bottom). HMGB1, WNT5B, C-MYC, β-catenin, and IL-6 are interacting proteins of MAX in CCA To define the MAX interactome, we performed immunoprecipitation (IP) with an anti-MAX antibody using liver lysates from CCA and saline control mice. Proteins were identified using mass spectrometry ( Figure 3A).
Meanwhile, C-MYC and CTNNB1 were the most relevant MAX-interacting proteins in "proteoglycans in cancer" pathways in CCA, with no reference in normal tissues (Supplemental Figure S10D, http://links.lww.com/HC9/A359). Some proteins such as Bcl9, TBC1d2b, and kifap3 also had higher scores as MAX-interacting proteins but were not related to cancer pathways. Hence, HMGB1, WNT5B, C-MYC, β-catenin, and IL-6 were considered as possible important MAX-interacting proteins in CCA by bioinformatics analysis. Furthermore, analysis of these partners in GEPIA human CCA database showed that IL-6, CTNNB1, HMGB1, and C-MYC exhibited a positive correlation with MAX mRNA levels (Supplemental Figure S10E-H, http:// links.lww.com/HC9/A359).
To validate these novel MAX interactors, we performed Co-IP with an anti-MAX antibody followed by western blotting from CCA and normal mouse liver lysates. HMGB1, β-catenin, and C-MYC interacted with MAX in the cytoplasm and nucleus ( Figure 3B). We also performed Co-IP with human CCA and normal human liver lysates and found higher levels of MAX interaction and more interaction with β-catenin in the cancer tissues ( Figure 3C). We next used recombinant MAX, β-catenin, and specific antibodies immobilized to A/G beads and found MAX and β-catenin can interact directly ( Figure 3D). We also found that DEN treatment increased HMGB1, β-catenin, C-MYC, and IL-6 protein expression ( Figure 3E) and mRNA levels ( Figure 3F) compared with control.

Cell-specific expression of MAX and MAXinteracting proteins in liver tissues with CCA
To determine the expression levels of MAX and MAXinteracting proteins, hepatocytes, KCs, HSCs, and cholangiocytes were isolated from liver with CCA and normal control. We found that the protein expression of MAX and HMGB1, β-catenin, C-MYC, and IL-6 was elevated in cholangiocytes from CCA tissue compared with control ( Figure 4A). We further isolated hepatocytes from mouse control and CCA livers and assessed the expression of MAX and its interacting proteins. The protein expression of HMGB1, β-catenin, C-MYC, and IL-6 in CCA-derived hepatocytes was 2.67-, 2.48-, 1.86-, and 2.58-fold, respectively, compared with control group ( Figure 4B). HMGB1 in KCs isolated from CCA liver tissues was 2-fold higher than controls ( Figure 4C, left). Our results also showed that the protein expression of β-catenin was 1.64-fold higher in the CCA-derived KCs compared with the control group ( Figure 4C, left). IL-6 expression in KCs isolated from CCA livers was 2.0-fold higher than that of control mice.
It is well known that activated HSCs promote cancer cell progression through paracrine or autocrine IL-6 signaling. [22] Our results showed that IL-6 levels were increased 1.55-fold in HSCs isolated from CCA liver tissues compared with the control group. HMGB1 expression in HSCs isolated from CCA liver tissues was increased 1.82-fold, while C-MYC and MAX were increased 2.24 and 1.85-fold, F I G U R E 2 Expression and stability of MAX and its role in MzChA-1 growth, migration, invasion, and tumorigenicity. (A) MAX strongly stained in the proliferated bile duct of primary sclerosing cholangitis (PSC) and cholangiocarcinoma (CCA). H&E stains are shown in the top (×10), and IHCs are shown in the bottom (×20). Local magnification of boxed areas is shown right for each IHC. IHC representative pictures are shown from n = 3. (B) MAX mRNA levels in NHL, CCA and PSC from 2-way ANOVA assay. Results are mean % ± SEM of NHL. † † †p < 0.001, ****p < 0.0001 vs. NHL. (C) MAX protein level in cytoplasm and nucleus from NHL and CCA was measured by Western blotting (n = 3 each) were normalized to housekeeping control β-actin (cytoplasm) or nucleus (Histone H3). Densitometric values are summarized below the blots, expressed as mean% of normal liver ± SEM. **p < 0.01, ***P < 0.001 vs. normal liver. (D) MzChA-1 and cholangiocytes isolated from mouse liver tissues with CCA and control livers were cultured at 24 hours, and then CHX treatment and Western blotting (top) were conducted. The MAX band intensity was normalized to β-actin and then normalized to the t = 0 controls. respectively, in the CCA group compared with controls ( Figure 4C, right). Cholangiocytes derived from CCA liver exhibited increased interaction of HMGB1, β-catenin, and C-MYC with MAX compared with control cholangiocytes ( Figure 4D). Furthermore, immunofluorescence showed that the expression of HMGB1, C-MYC, and β-catenin in cholangiocytes from CCA liver was higher than normal liver in the cytoplasm and nucleus while IL-6 increased just in cytoplasm ( Figure 4E).

CSNK2A1-mediated MAX phosphorylation increased C-MYC and β-catenin binding and regulated HMGB1 promoter activity through E-BOX
CK2α inhibits the DNA-binding activity of MAX-MAX homodimers but not MYC-MAX heterodimers. [23] CK2/ CSNK2A1 is involved in cancer progression by phosphorylating various signaling molecules. [9] We therefore hypothesized that the switch of MAX-MAX homodimerization to MYC-MAX or β-catenin-MAX heterodimerization may regulate HMGB1 promoter activity through the E-box. MAX contains one important canonical SXXE/D motif of CK2 [24] at S11. CSNK2A1 was predicted to bind the SXXE/D motif of MAX at the S11 phospho-site ( Figure 5A). We also found that CSNK2A1 and MAX S11 expression was increased in cholangiocytes from liver tissue with CCA compared with controls ( Figure 5B). Available CCA data sets showed that CSNK2A1 mRNA levels are higher in iCCA compared with adjacent nontumor liver tissue (GSE76297) (Supplemental Figure S8F, http://links.lww.com/HC9/A357) and iCCA compared with normal human biliary epithelial cells (GSE32225) (Supplemental Figure S8G, http://links. lww.com/HC9/A357). Moreover, the GEPIA database shows that CSNK2A1 mRNA significantly increases in CCA compared with normal liver tissues (Supplemental Figure S8D, Compared with MzChA-1 cells that had high CSNK2A1 levels, H69 cells have very low CSKN2A1 expression ( Figure 5G). We found that the expression of HMGB1, β-catenin, C-MYC, and IL-6 in H69 cell line could not be altered by overexpressing or silencing MAX ( Figure 5H, I). The overall data indicate that MAX's effects on C-MYC, β-catenin, HMGB1, and IL-6 may be dependent on the cellular level of CSNK2A1.
To further assess the role of CSNK2A in regulating MAX's function, we used the specific and selective CK2 ATP competitive inhibitor (CX-4945, Silmitasertib) that has been administered in human trials as an anticancer drug. We found that CX-4945 could inhibit mRNA and protein expression of basal and MAX OV-induced C-MYC, β-catenin, HMGB1, and IL-6 in MzChA-1 ( Figure 5J, K). Since there is low expression of CSNK2A1 and phospho-max in H69 ( Figure 5G), it is possible that CX-4945 may not work there in inhibiting MAX targets (Supplemental Figure S10E, F, http://links. lww.com/HC9/A359).

MAX phosphorylation is involved in upregulation of HMGB1 and IL-6 promoter activity
The HMGB1 promoter region (−298/+1) (Supplemental Figure S12, http://links.lww.com/HC9/A361) was assessed for its responsiveness to MAX overexpression in MzChA-1 cells. MAX knockdown lowered HMGB1 promoter-driven luciferase activity, whereas MAX OV had the reverse effect ( Figure 6A, B). To evaluate whether the effect of MAX on the HMGB1 promoter was through E-box region, we mutated the E-box-binding site in the promoter. Mutation at the E-box-binding site lowered HMGB1 promoter activity by about 50% (Figure 6A, B). CX-4945 decreased HMGB1 promoter activity, but this was attenuated with E-box mutation ( Figure 6A, B). To determine whether phosphorylation of MAX is involved in HMGB1 promoter activities, ChIP and qPCR analysis were performed by spanning the E-box region in MzChA-1 cells using MAX and phospho S11 MAX antibodies. ChIP assays showed MAX knockdown lowered MAX binding to E-box region of the HMGB1 promoter. MAX overexpression increased MAX binding to E-Box region. There was more binding to the E-box in phosphorous S11 MAX compared with nonphosphorous MAX. Interestingly, CX-4945 treatments enhanced the effect of MAX siRNA knockdown and overexpression ( Figure 6C). It explains MAX phosphorylation mediates HMGB1 promoter activities.
MAX phosphorylation may increase combination of phosphorylated MAX with C-MYC or β-catenin. Seq-ChIP and qPCR analysis was performed using C-MYC antibody after ChIP of MAX and phospho S11 MAX antibodies. Results showed that MAX knockdown or overexpress lowered or increased C-MYC binding to E-box region of the HMGB1 promoter, respectively. Furthermore, there was more binding to the E-box in phosphorous S11 MAX compared with nonphosphorous MAX. Moreover, CX-4945 treatments enhanced the effect of MAX siRNA knockdown and overexpression ( Figure 6D, E). Immunofluorescence in cholangiocytes after MAX OV treatments showed that MAX staining (including antibodies of MAX and phospho S11 MAX) increases in cytoplasm and nucleus. Phospho-MAX staining of nucleus increases markedly in MAX OV+ phosphor S11 MAX antibody compared to MAX OV+ MAX antibody. CX-4945 treatment inhibits nuclear immunofluorescence staining of MAX ( Figure 6F). Intrigued by the MAX-, C-MYC-, and β-cateninmediated regulation of HMGB1 reporter activity, we examined whether MAX, C-MYC, and β-catenin can bind to the E-box element using nuclear proteins from H69, normal human liver, MzChA-1, and human CCA liver. Figure 6G shows that MAX, C-MYC, and β-catenin can bind to the E-box element (Shift bands, Figure 6G). Interestingly, β-catenin can heterodimerize with MAX to bind to the E-box element ( Figure 6G). This suggests that MAX's inductive effect may be, in part, by increasing β-catenin's expression and binding to the E-box element. Meanwhile, these results suggested that phospho S11 MAX may positively regulate the promoter activities by interacting with and enhancing the binding of C-MYC and β-catenin to the E-box element, which positively regulated HMGB1 transcription.
Furthermore, we also assessed IL-6 promoter activities using MAX knockdown and overexpression in MzChA-1 cells. MAX knockdown and overexpression decreased and increased IL-6 promoter-driven luciferase activity, respectively ( Figure 7A, B). To explore the regulatory site of MAX on the IL-6 promoter, we mutated the TCFs/LEF1-binding site in the IL-6 promoter. Mutations in the TCFs/LEF1binding site and CX-4945 both reduced IL-6 promoter activity. Interestingly, the effect of CX-4945 on reducing IL-6 promoter activity was attenuated by TCFs/LEF1 mutations ( Figure 7A, B). However, it was uncertain whether the phosphorylation of MAX was involved in the regulation of IL-6 promoter activity. We performed Seq-ChIP assays using MAX and phosphor S11 MAX antibodies after βcatenin ChIP by spanning the TCFs/LEF1 region in MzChA-1 cells. The results showed that MAX knockdown and CX-4945 reduced the binding of β-catenin binding to the TCFs/LEF1 region of the IL-6 promoter, whereas MAX OV had the opposite effect. CX-4945 reduced MAX OVmediated IL-6 promoter activities ( Figure 7C). To determine whether MAX phosphorylation increases β-catenin binding to TEFs/LCF1 site, ChIP, Seq-ChIP and qPCR were performed using MAX and phospho S11 MAX antibodies. Phospho-MAX staining of nucleus increases markedly in MAX OV+ phosphor S11 MAX antibody compared to MAX OV+ MAX antibody. CX-4945 treatment inhibits nuclear immunofluorescence staining of MAX ( Figure 6F). The results showed that MAX knockdown or overexpression decreased or increased the binding of MAX to the IL-6 promoter TCFs/LEF1 region, respectively. In addition, there was more binding to the E-box in phosphorus S11 MAX compared with nonphosphorus MAX. CX-4945 treatment enhanced the effect of MAX siRNA knockdown and overexpression ( Figure 7D). Impressed by the MAX and β-catenin-mediated regulation of IL-6 reporter activity, we examined whether MAX and β-catenin can bind to the TEFs/LCF1 element using nuclear proteins from H69, normal human liver, MzChA-1, and human CCA liver. Figure 7E shows that MAX and β-catenin can bind to the TEFs/LCF1 element (Shift bands, Figure 7E). Even though IL-6 promoter does not have E-box sites, β-catenin can heterodimerize with MAX to bind to the TCFs/LCF1 element ( Figure 7E). This suggests that MAX's inductive effect may increase β-catenin's expression and bind to the E-box element. Meanwhile, these results suggested that phospho S11 MAX may positively regulate the promoter activities by interacting with and enhancing the binding of βcatenin to the TCFs/LCF1 element, which positively regulated IL-6 transcription.

MAX promotes HMGB1 and IL-6 through paracrine and autocrine mechanisms
To determine the role of overexpressed MAX on HMGB1 and IL-6 secretion from HepG2, LX2, MzChA-1, and RAW cells, we first analyzed the protein expression of HMGB1 and IL-6 in the cell culture medium. Of the 4 cell types examined, overexpression of MAX increased IL-6 release maximally from RAW cells, whereas HMGB1 release was the highest in LX2 cells ( Figure 8A). Next, we investigated whether MAX overexpression in HepG2, LX2, and RAW cells promoted cell growth, migration, and invasion of MzChA-1 cells. We found that coculturing MzChA-1 with MAX overexpressing HepG2, LX2, or RAW cells markedly promoted cell growth ( Figure 8B), migration ( Figure 8C), and invasion ( Figure 8D) compared with control (empty vector). Furthermore, media from HepG2, LX2, and RAW cell overexpressing IL-6 ( Figure 8E) and HMGB1 ( Figure 8F) measurably promoted MzChA-1 cell migration. In contrast, silencing of IL-6 ( Figure 8E) or HMGB1 ( Figure 8F) inhibited MzChA-1 cell migration.

Effects of CX-4945 on CCA cell growth and in vivo tumorigenicity
To determine the effects of CX-4945 on MzChA-1 cell proliferation and invasion, we treated the cells at 10μM and examined the effect on cell growth. After 24h of treatment, CX-4945 reduced proliferation to 32% as compared with DMSO control group ( Figure 8G). CX-4945 at 10 µM significantly reduced cell invasion to 39% as compared with DMSO control group ( Figure 8H). Meanwhile, CX-4945 significantly inhibited cell migration ( Figure 8I). To confirm their effects in vivo, the orthotopic tumorigenicity used injection of MzChA-1 cells into the left hepatic lobe as described. [21] From day 3, these mice were treated with CX-4945 or a vehicle orally at 100 mg/kg twice daily for 27 days. The CX-4945-treated mice reduced tumor site to 67% ( Figure 8J). Interestingly, CX-4945 treatment lowered HMGB1, β-catenin, C-MYC, phosphor-MAX, IL-6, and CSNK2A1 protein levels in expression is upregulated in cholangiocytes, KCs, HSCs, and hepatocytes. Furthermore, MAX positively regulates IL-6 expression. Induction of MAX in HepG2, LX2, and RAW cells promoted migration of MzChA-1 cells, and silencing IL-6 or HMGB1 prevented MAX from exerting its positive effect on migration. These data strongly support the role of IL-6 and HMGB1 as downstream effectors of MAX. CX-4945 also lowers IL-6 expression and inhibits CCA cell growth, invasion, and migration in vitro and tumorigenesis in vivo. The results suggest that CX-4945 could be of therapeutic relevance in CCA through IL-6 downregulation.
Recent literature studies show that β-catenin signaling drives widespread gene repression and activation. [34] We found that β-catenin expression in liver tissues during DEN treatment increased from week 1 onward. β-catenin protein expression was increased in cholangiocytes, KCs, HSCs, and hepatocytes. Phosphorylation of MAX inhibits the DNA-binding activity of MAX homodimers but not C-MYC/MAX heterodimers. [24,28] On the basis of the interaction between β-catenin, C-MYC, and MAX and their co-occupancy on the HMGB1 promoter E-box, we believe that β-catenin-MAX may form heterodimers together with C-MYC-MAX heterodimers to regulate HMGB1 and IL-6 expression.
In conclusion, we have established a novel model of CCA progression from biliary proliferation to atypical cystic hyperplasia and to, eventually, cholangiocarcinogenesis. MAX acts as a master regulator through the upregulation of HMGB1, IL-6, C-MYC, and β-catenin in hepatic cells from liver tissues with CCA. C-MYC-MAX and β-catenin-MAX binding to E-box site or β-catenin-MAX bound to TCFs/LEF1 enhanced HMGB1 or IL-6 promoter activities. IL-6 and HMGB1 secreted by hepatocytes, HSCs, and KCs exert paracrine effects on cholangiocytes to promote cell growth, migration, and invasion and lead to the progression of cholangiocarcinogenesis. IL-6 and HMGB1 are downstream effectors of MAX. The combination of CSNK2A1 and MAX at S11 can regulate the occurrence of this series of processes. We propose that CX-4945 and MAX-interacting proteins provide perspectives on therapeutic strategies to attenuate progression from atypical cystic hyperplasia to cholangiocarcinogenesis.

Statement of ethics
All animals were recruited in accordance with US law and institutional ethical guidelines. Samples were obtained from Cedars-Sinai Medical Center. The study protocol was approved by the US government and ethics committee.

AUTHOR CONTRIBUTIONS
Bing Yang, Heping Yang, and Jing Zhang reviewed the literature, drafted the manuscript, and performed data collection; Bing Yang performed data collection, analysis, and interpretation, as well as figure preparation; Jiaohong Wang performed cell culture, analysis, and interpretation; Wei Fan assisted in the literature review and drafted the manuscript; Monica Anne R. Justo, Lucía Barbier-Torres, Justin Steggerda, Shelly C. Lu, Yongheng Chen, Komal Ramani, and Shelly C. Lu provided critical reading and editing of the manuscript; and Xi Yang and Ting Liu performed data collection and analysis. All authors have read and approved the final manuscript.

FUNDING INFORMATION
This work was supported by NIH grants P01CA172086 (Heping Yang, and Shelly C. Lu), R01DK107288 (Heping Yang and Shelly C. Lu) and National Natural Science Foundation of China (82070632, Ting Liu). The funders had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation.